Summary: Engagement of multicomponent immunoreceptors such as the T cell antigen receptor results in rapid recruitment and activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate a number of enzymes and adapter molecules involved in complex signaling cascades. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane protein. LAT is a critical transmembrane adapter protein. We have performed studies to characterize how LAT is phosphorylated and binds a number of critical signaling molecules, thus bringing other adapter molecules and enzymes in multimolecular complexes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, microscopic and genetic techniques are currently employed to study the characteristics of LAT-based signaling complexes and the enzyme pathways that are coupled to and activated at LAT complexes. In the past year we have published three studies that can be viewed as a continuation of previous work. The results of many years of work on the LAT molecule was reviewed in this period. This manuscript focused on our studies looking at LAT-based complexes at molecular, nanostructure and microscluster size scales. In particular, using purified proteins to form complexes in vitro resulted in insights into the cooperativity of protein binding and the role of oligomerization of signaling complexes. These observations were followed by microscopic and functional studies. Use of superresolution microscopy led to insights in the nanostructure of these complexes in cells. In turn nanoclusters coalesce into microclusters, which have been carefully studied by confocal microscopy in this laboratory over the past fifteen years. The review article summarized the many studies and insights obtained over this period. A new study in which a form of super-resolution microscopy, photo activation localization microscopy (PALM), was used extended our studies of signaling complexes. In a 2011 study we performed two-color microscopy with this technique and demonstrated the nanostructure of complexes in cells. In the new work, we developed a novel method of using three photoactivable reagents to enable simultaneous analysis of three different molecules in fixed cells. Statistical techniques were developed to analyze structure and synergistic interactions between molecules. We provided additional information about protein-protein interactions in signaling complexes. Our laboratory previously reported on LAT dynamics and the effect of ubiquitinylation-defective LAT mutant molecules. In T cell lines expression of these mutants enhanced T cell activation, and several in vivo functions such as cytotoxic killing and antibody production were positively affected. However despite these findings, greater T cell effector function expression mediated by the mutant LAT molecules did not have an effect on clearance of certain pathogens or tumors. These studies on LAT mutants led us to a collaboration with the Restifo laboratory in CCR, NCI. Investigators in that program had demonstrated that mice lacking the Cish molecule, a member of the SOCS family of ubiquitin E3 ligases, showed enhanced clearance of tumors. They had evidence that the effect was mediated by enhanced T cell signaling. In our collaboration we were able to demonstrate that a physiological target of Cish is the enzyme, phosopholipase C-gamma 1 (PLC-g1). In the presence of Cish PLC-g1 is targeted for degradation through ubiquitin-mediated degradation. Without Cish enhanced PLC-g1 signaling mediates enhanced T cell function. Pharmacologic targeting of Cish might prove useful in enhancing immunotherapy regimens.